Infections caused by Enterobacteriaceae, primarily the Gram-negative pathogens Klebsiella pneumoniae and Escherichia coli, are becoming increasingly difficult to treat owing to widespread resistance to several classes of antibiotics including penicillins, cephalosporins, carbapenems, fluoroquinolones and antifolates. Along with resistance, the naturally limited array of agents effective against Gram-negative pathogens and the dearth of antibiotic discovery in the pharmaceutical industry combine to create a critical need for new drug discovery. For the past several years, we have used a structure-based effort to develop a novel series of propargyl-linked antifolates that potently inhibit the essential enzyme dihydrofolate reductase (DHFR) and are effective against Gram-positive and eukaryotic pathogens. Additionally, these compounds show low rates of resistance and have good physicochemical properties. Recently, we have discovered that the propargyl-linked antifolates are potent inhibitors of K. pneumoniae in culture and against K. pneumoniae DHFR. Here, we propose to extend this class of antifolates to become excellent antibiotics against pathogenic Enterobacteriaceae. We propose three specific aims. In the first aim, we will develop inhibitors that are potent and selective inhibitors of K. pneumoniae and E. coli DHFR and potent inhibitors of wild-type and resistant Enterobacteriaceae, such as trimethoprim-, ESBL-, KPC- and NDM1-variants while maintaining low human cell toxicity. In the second aim, we will determine iterative crystal structures of wild- type and trimethoprim-resistant Enterobacteriaceae DHFRs as well as human DHFR, intended to drive the design of potent and selective compounds. The third aim will focus on studies in animals: an initial stage with a set of potent compounds begins with the evaluation of efficacy against wild-type strains and initial pharmacokinetic parameters. A second stage will evaluate efficacy against a range of phenotypes along with detailed pharmacokinetic/pharmacodynamic parameters. At the end of this proposal we expect to deliver a highly efficacious, orally available antifolate antibiotic against a broad range of Enterobacteriaceae isolates.